This application addresses broad Challenge Area (04) Clinical Research and specific Challenge Topic (04-HL- 103): Assess the role of leukocyte interactions with platelets, erythrocytes, and endothelium in the pathogenesis of heart, lung, and blood diseases. The purpose of the proposed studies is to identify a key point in the communication between leukocytes and platelets that can serve as a target for novel pharmaceuticals that will prevent vascular occlusion despite vessel injury. Cardiovascular diseases, including coronary heart disease, stroke, and peripheral arterial disease, are the leading cause of morbidity and mortality in the US. Antiplatelet therapy with aspirin, clopidogrel, or their combination, is currently standard of care for prevention of myocardial infarction (MI), stroke, and following percutaneous coronary intervention. Although effective, many individuals suffer vascular occlusive disease events despite these therapies. Greater blood neutrophil counts are consistently associated with MI and stroke in primary and secondary prevention cohorts, and recent data demonstrate an association between higher neutrophil counts and resistance to the effects of aspirin and clopidogrel. Greater neutrophil counts are also associated with the extent of myocardial and neuronal damage after MI and stroke, respectively. Despite these clinical observations, a mechanism linking neutrophils to cardiovascular morbidity has not been identified. We hypothesize that neutrophils function as physiologic regulators of platelet activation and vascular occlusion at sites of vessel injury in vivo, and that neutrophil cathepsin G is a key molecular mediator of these effects. We will translate observations from human clinical studies into 3 distinct models in mice to test our hypotheses: tail bleeding time, ferric chloride-induced injury of mesenteric arterioles, and transient filament occlusion of the middle cerebral artery. We will examine the effects of neutrophil count and cathepsin G on vascular outcomes by manipulating neutrophil counts in vivo, using a specific pharmacologic inhibitor of cathepsin G, and using cathepsin G knockout mice. At the end of the 2-year study period we expect to demonstrate that: 1) neutrophilia promotes platelet activation and vascular occlusion in mouse models of vascular injury;2) neutrophil cathepsin G is a critical molecular mediator of platelet accumulation and luminal occlusion at sites of vascular injury;and, 3) neutrophilia promotes resistance to the anti-platelet effects of aspirin in vivo but does not reduce the vaso-protective effects of cathepsin G inhibition. Johns Hopkins is one of the biggest employers in the state of Maryland, generating ~$10 billion in economic activity annually. Funds from the American Recovery and Reinvestment Act will allow us to employ the research team, creating or preserving 5 jobs, required to reach our scientific goals in a 2-year timeframe. By doing so, we expect to demonstrate that cathepsin G is a physiologically relevant target for prevention and treatment of inflammation-related vascular occlusive disorders such as MI and stroke. PUBLIC HEALTH RELEVANCE: The purpose of the proposed studies is to identify a key point in the communication between leukocytes and platelets that can serve as a target for novel pharmaceuticals that will prevent vascular occlusion despite vessel injury. We hypothesize that neutrophils function as physiologic regulators of platelet activation and vascular occlusion at sites of vessel injury in vivo, and that neutrophil cathepsin G is a key molecular mediator of these effects. We will examine the effects of neutrophil count and cathepsin G on vascular outcomes by manipulating neutrophil counts in vivo, using a specific pharmacologic inhibitor of cathepsin G, and using cathepsin G knockout mice. At the end of the 2-year study period we expect to demonstrate that: 1) neutrophilia promotes platelet activation and vascular occlusion in mouse models of bleeding time, arteriolar thrombosis, and stroke;2) neutrophil cathepsin G is a critical molecular mediator of platelet accumulation and luminal occlusion at sites of vascular injury;and, 3) neutrophilia promotes resistance to the anti-platelet effects of aspirin in vivo but does not reduce the vaso-protective effects of cathepsin G inhibition.